DNA methylation is an epigenetic modification that regulates gene expression and marks imprinted genes. Consequently, aberrant DNA methylation is known to disrupt embryonic development and cell cycle regulation, and it can promote oncogenesis that produces cancers. In mammals, methylation occurs only at cytosine residues and more specifically only on a cytosine residue that is adjacent to a guanosine residue (that is, at the sequence CG, often denoted “CpG”). Detecting and mapping sites of DNA methylation are essential steps for understanding epigenetic gene regulation and providing diagnostic tools for identifying cancers and other disease states associated with errors in gene regulation.
Mapping methylation sites is currently accomplished by the bisulfite method described by Frommer, et al. for the detection of 5-methylcytosines in DNA (Proc. Natl. Acad. Sci. USA 89: 1827-31 (1992), explicitly incorporated herein by reference in its entirety for all purposes) or variations thereof. The bisulfite method of mapping 5-methylcytosines is based on the observation that cytosine, but not 5-methylcytosine, reacts with hydrogen sulfite ion (also known as bisulfite). The reaction is usually performed according to the following steps: first, cytosine reacts with hydrogen sulfite to form a sulfonated cytosine. Next, spontaneous deamination of the sulfonated reaction intermediate results in a sulfonated uracil. Finally, the sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because uracil forms base pairs with adenine (thus behaving like thymine), whereas 5-methylcytosine base pairs with guanine (thus behaving like cytosine). This makes the discrimination of methylated cytosines from non-methylated cytosines possible by, e.g., bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16: 431-36; Grigg G, DNA Seq. (1996) 6: 189-98) or methylation-specific PCR (MSP) as is disclosed, e.g., in U.S. Pat. No. 5,786,146.
A gene's methylation state or mutation/polymorphism state is often expressed as the fraction or percentage of individual strands of DNA that are methylated/mutant at a particular site (e.g., at a single nucleotide or at a longer sequence of interest, e.g., up to a ˜100-bp subsequence of a DNA) relative to the total population of DNA in the sample comprising that particular site. For simplicity, the discussion below is directed to measuring methylation but it is equally applicable to the measurement of mutations and polymorphism in nucleic acid populations.
Traditionally, the amount of unmethylated (e.g., native) gene is determined by quantitative PCR (qPCR) using calibrators. Then, a known amount of DNA is bisulfite treated and the resulting methylation-specific sequence is determined using either a real-time PCR or an equivalent exponential amplification. In particular, conventional methods generally comprise generating a standard curve for the unmethylated target by using external standards. The standard curve is constructed from at least two points and relates the real-time Cp value for unmethylated DNA to known quantitative standards. Then, a second standard curve for the methylated target is constructed from at least two points and external standards. This second standard curve relates the Cp for methylated DNA to known quantitative standards. Next, the test sample Cp values are determined for the methylated and unmethylated populations and the genomic equivalents of DNA are calculated from the standard curves produced by the first two steps. The percentage of methylation at the site of interest is calculated from the amount of methylated DNAs relative to the total amount of DNAs in the population, e.g., (number of methylated DNAs)/(the number of methylated DNAs+number of unmethylated DNAs)×100.
Accordingly, these conventional methods require the construction of standard curves from several external standard PCRs and then require calculating a putative absolute number of methylated DNA sites or strands in one portion of the test sample and a putative absolute number of unmethylated sites or strands of DNA from another portion of the test sample. These methods require the user to assemble several reaction mixtures, which can be labor intensive and time-inefficient, and which increases the likelihood of error. In addition, the number of reactions requires a relatively large amount of DNA to provide enough template for all the necessary PCR mixtures, and thus is sample-inefficient. Furthermore, each of the numerous measurements has an associated error that is propagated in calculating the extent of methylation in the test sample. In particular, at least two standards are assembled and measured to construct the methylated DNA standard curve, at least two standards are assembled and measured to construct the unmethylated DNA standard curve, and multiple aliquots of the test sample are assembled and measured. Additionally, well-to-well variation (e.g., amongst the wells of a 96-well assay plate) between external standards and the test sample can also introduce significant errors in the measurement. For instance, the typical calibration methods used for fluorescence real-time PCR thermocyclers can unpredictably produce well-to-well variations of 1 Cp unit or more. As such, these variations in sample measurement as a function of location on the assay plate can cause substantial errors for the analysis of a test sample.